The objective of the proposed NMR studies is to determine the protein-protein interactions involved in the assembly of a bacterial nanoinjector that is essential in the pathogenesis of many bacterial pathogens. The nanoinjector is a needle-like macromolecular assembly and forms the structural part of bacterial protein export machinery, the type III secretion system (T3SS). The T3SS also consists of effector proteins, which are injected into the host cell cytoplasm, and chaperones, which form complexes with T3SS proteins while inside the bacterial cytoplasm. The T3SS needle apparatus is assembled from over 20 different proteins and consists of a basal structure anchored on the bacterial membranes, the needle itself on the bacterial surface, a tip complex, and a translocon. The translocon is the membrane spanning structure that punctures a hole on the host cell membrane to allow the passage of bacterial proteins into the host cell cytoplasm. On the first round of funding, we have determined how the needle protein interacts with the tip protein. Currently, how the tip protein interacts with the translocon is completely unknown. For this renewal, our Aim #1 is to determine how the tip protein interacts with the translocon proteins. Another unknown is how the tip proteins interact with their chaperones, thus our Aim #2 is to determine how the tip proteins interact with their chaperones. To advance our understanding of the biology of the T3SS, our NMR studies will be complemented with electron microscopy and functional assays. Many pathogens rely on the type III secretion system to infect millions of people worldwide. Because the needle apparatus is exposed on the bacterial surface, disrupting the needle assembly is an attractive target for the development of novel anti- infectives. This approach requires a detailed understanding of the protein-protein interactions involved in needle assembly.